Plant Biology

Abstract Tricyclic coumarins (TCs) exhibit defensive properties against herbivorous insects and possess valuable pharmacological properties for humans. They are mainly distributed in the Apiaceae, Rutaceae, Moraceae, and Fabaceae families. The chemical structural diversity of TCs in these families primarily arises from differences in skeletal variety and attachment position of the third ring, as observed in unsaturated furanocoumarins (FCs) and saturated dihydrofuranocoumarins (DHFCs). In TC biosynthesis, the FC and DHFC pathways diverge through distinct oxidation patterns. The product specificity of the reported cytochrome P450 monooxygenase at the branching point suggests that these TC groups are produced in nearly identical ratios; however, some species of the genus Angelica (Apiaceae) accumulate DHFCs at higher levels than FCs. In this study, we investigated the enzymatic mechanism responsible for forming these two TC groups using A. keiskei, a Japanese native species used as a medicinal and edible herb. Organ-specific transcriptome analysis highlighted a P450 member CYP71AJ62 that exhibited a close correlation with TC accumulation across organs. Enzymatic characterization demonstrated that CYP71AJ62 predominantly forms a hydroxylated DHFC, vaginidiol, which may promote DHFC over FC skeletal formation in TC biosynthesis. Phylogenetic analysis suggested that CYP71AJ62 has been derived from a gene responsible for FC formation by functional shift without gene duplication. Subsequent three-dimensional structural modeling and site-directed mutagenesis revealed that two amino acid residues on substrate recognition sites cooperatively determine the product specificity of this P450 enzyme. This study provides new insights into the molecular evolutionary process underlying ring-structure diversification and, more broadly, the adaptation of plants to their environments.

Fire-dependent or fire-influenced ecosystems harbor species with diverse response strategies, such as smoke-stimulated germination and dormancy release through heat shocks. Although these cues influence germination in many fire-prone ecosystems, their effects on seeds of woody species from tropical dry forests (TDFs) remain poorly understood. We evaluated the germination response of twelve TDF woody species to three heat-shock temperatures (80, 120, and 160C), two exposure times (3 and 5 min), and two smoke conditions (with and without smoke water), plus dry and wet controls. We also assessed the relationship between germination and functional seed traits, including permeability, seed-coat thickness, mass, and shape index. Of the eleven species that germinated, four showed broad tolerance, with no significant reduction in germination even at 160C for 5 min, which was associated with lower permeability, thicker seed coats, and greater mass. Four other species showed restricted tolerance to heat, with significant declines in germination at higher temperatures or longer exposure times, and were associated with higher permeability, thinner seed coats, and lower seed mass. The remaining three species showed heat-stimulated germination between 80 and 120C and tended to have lower shape-index values. Smoke treatments significantly increased germination in three species, Ceiba pentandra, Guazuma ulmifolia, and Samanea saman. In the remaining species, significant differences were either absent or attributable to moisture or imbibition effects rather than to smoke itself. Overall, germination responses to heat were associated with variation in functional seed traits, whereas responses to smoke were less widespread and showed a less consistent functional pattern.

Seed ecophysiology is essential for understanding plant regeneration and developing effective conservation and ecological restoration strategies, yet knowledge remains scarce for most threatened Cerrado species. We characterised the germination niche, desiccation tolerance and storage behaviour of Xyris paradisiaca (Xyridaceae), an Endangered species endemic to Central Brazil, to assess whether its restricted distribution is associated with narrow germination requirements and to inform seed-based conservation. Germination was tested across light regimes, constant temperatures, decreasing water potentials and short-duration heat shocks, while thermal- and hydro-time models were used to quantify thermal and hydric thresholds. Seeds exhibited an absolute light requirement but broad abiotic tolerances. Germination remained >87% between 15 and 40 {degrees}C, with estimated cardinal temperatures of Tb = 8.94 {degrees}C, To = 33.13 {degrees}C and Tc = 45.53 {degrees}C. Germination remained similar to the control down to -0.6 MPa and exceeded 40% at -1.0 MPa, with a median base water potential of {Psi}b = -1.03 MPa. Germination also remained high after 1-min heat shocks up to 200 {degrees}C. Seeds were desiccation tolerant, with 100% germination after drying and a viability loss index of -0.04, and germination remained >90% after 24 months of ambient storage. Thus, the highly restricted distribution of X. paradisiaca is not driven by a narrow physiological germination niche. Its broad environmental tolerances, desiccation tolerance and favourable storage behaviour also highlight its unexplored potential for seed-based restoration, propagation and ex situ conservation.

Distinct from plants which directly parasitise other plants (parasites), non-photosynthetic mycoheterophic plants (MHPs, previously known as achlorophyllous mycotrophs or saprophytes) depend upon fungal hyphae for their carbon and have been viewed as cheaters or parasites in their fungal partnerships rather than mutualistic as in most other plant-mycorrhizal interactions. Over 400 such species in 87 genera and 11 families are recorded which maintain this lifeform through their life-cycle, while an estimate 30,000 further species, mainly orchids, depend on this mode of life to establish themselves from seeds. In most cases the fungal partners and the ultimate carbon sources, usually autotrophic plants, are unknown. High fungal specificity is the norm in those MHPs researched. We describe a new MHP genus and species, Denisea fonensis that we discovered in the Simandou range of the Republic of Guinea, West Africa in October 2025 on the footprint of a mining project. While clearly placed in Dioscoreales (which has three MHP families), the features of Denisea are so unusual that it was not initially clear in which family to place it. Finally, with molecular data we placed Denisea in Thismiaceae, corroborated by morphology. The new discovery is all the more unexpected as this is the first record of Thismiaceae from West Africa. Until now the family has only been recorded in Africa from three species of the very different genus Oxygyne, of Cameroon and Central African Republic. We provisionally assess Denisea fonensis as Critically Endangered since less than ten individuals are recorded in only a few square metres at one site, where there are imminent threats from animals. We propose urgent measures to conserve this species to reduce the risk of global extinction that has eliminated other African MHP species such as Oxygyne triandra, and further research to determine the fungal and autotrophic plant partners of this remarkable plant genus.

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.

To elucidate the resistance of hybrid larch (Larix spp.) clones to Dendrolimus superans and their chemical defense mechanisms, 46 clones were subjected to field feeding-infestation experiments. Insect resistance was comprehensively evaluated by the membership function method from three perspectives: insect weight change, frass weight and defoliation rate (damage consequences); pre- and post-infestation physiological indices (constitutive and induced resistance). High- and low-resistance materials screened from each perspective were further analyzed by widely targeted LC-MS metabolomics. The three damage indices differed highly significantly among clones (P<0.01) and were positively correlated, indicating abundant genetic variation in resistance. Clones such as -30-C, -35-A and -21-A showed strong overall resistance, whereas -28-E and -20-A showed high constitutive and induced resistance. Defence enzyme activities (POD, SOD, CAT, PPO and PAL) correlated closely with secondary metabolites (total phenolics, tannins and total flavonoids) and nutrients, and rose synergistically after feeding, reflecting synergy between constitutive and induced resistance. In total, 3,577 metabolites were identified, predominantly terpenoids (>90%). The three groups yielded 1,010, 1,086 and 922 differentially accumulated metabolites, and PCA separated high- from low-resistance materials in all groups. These metabolites were significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, monoterpenoid and diterpenoid biosynthesis, and phenylpropanoid biosynthesis. In conclusion, resistance of hybrid larch to D. superans is jointly conferred by the defence enzyme system, secondary metabolite accumulation and a 'terpenoid-phenolic' metabolic network: terpenoids and phenolics act synergistically via the terpenoid and phenylpropanoid pathways, together forming the core of chemical defense and supporting the breeding of insect-resistant clones.

Drought and waterlogging are prominent climate change challenges that, alone or in sequence, pronouncedly constrain agricultural crop production. A better understanding of relationships between water availability and nutrition regarding plant functioning is needed to optimise fertilisation under these challenges. We investigated impacts of drought, waterlogging and drought followed by waterlogging under two fertilisation regimes (low, normal) on various traits of wheat (Triticum aestivum). Next to biomass and photosynthesis measurements, cross-platform metabolomics was applied for in-depth metabolic analyses of flag leaves. We found detrimental effects of drought and drought-waterlogging on shoot and root biomass, especially at normal fertilisation, while photosynthetic traits were not or only slightly affected. Foliar nitrogen contents were higher in plants exposed to drought or to drought-waterlogging. Numerous primary and specialised metabolites were modulated by fertilisation and/ or watering. In particular, amino acids, benzoxazinoids and flavonoids were affected by fertilisation. Due to watering especially several amino acids changed in concentrations, with the induction pattern partly depending on fertilisation. Our study deepens the understanding of the interplay between water and nutrient availabilities as well as nutritional demands, growth and metabolism of wheat. This is highly relevant to balance sustainable agricultural nutrient management under the challenges of ongoing climate change.

Sporadically, all plastids produce dynamic tubular extensions in response to myriad stresses. Although the use of fluorescent proteins revealed these transient tubules to be stroma-filled and led to their being called stromules, the mechanism underlying tubule extension from a spheroidal plastid remained unknown. As plastids in viridiplantae have two bounding membranes the fluid stroma remains confined within the inner envelope membrane (IEM) while the outer envelope membrane (OEM) around the IEM forms the interface with the rest of the cytoplasm. Here, using a sucrose treatment for inducing tubules in the dark in transgenic Arabidopsis plants expressing different stroma and OEM-targeted fluorescent proteins we carried out comparative molecular-genetic and lipidomics analyses. Our data strongly suggest that plastid tubule formation occurs due to increase in bilayer-forming galactolipids such as digalactosyldiacylglycerol (DGDG) on the OEM. While the dynamic behaviour of stromules occurs within an external encasing OEM, stresses such as phosphate starvation and high light intensity cause the galactolipid-rich OEM-tubule projecting beyond the stromule to be shed into the cytoplasm. OEM shedding from long tubules with extended plastid outreach into the cell suggests their important role in dispersing plastid-derived, membrane-stabilizing, galactolipids to the rest of the cell to augment its resilience to stress.

Bulk phloem flow is driven by pressure gradients generated by water drawn from the xylem, coupling source-sink transport with water status. Huanglongbing (HLB), a phloem-affecting bacterial disease of citrus, disrupts phloem transport, and causes canopy water deficit. Although improved water management can enhance HLB-affected tree health, the effects of HLB on water-driven source-sink relations are unknown. Fruits function as both carbon sink and water reservoir, but the role of fruit in regulating whole-tree water status remain poorly understood. We examined leaf-fruit water relations in HLB-affected citrus and evaluated how HLB exposure duration, fruit developmental stage, and source-sink manipulation shape these dynamics. Source-sink relations were manipulated in branches of symptomatic trees with long-term (four years) and recent (six months) HLB exposure. We girdled branches with a single fruit as the sink and defoliated at (0%, 50%, or 75%), and an ungirdled, non-defoliated branch served as control. We measured stomatal conductance, leaf hydraulic traits, leaf and fruit soluble sugars, diurnal fruit shrinkage (to estimate xylem backflow) and fruit water loss. Source-sink manipulation did not affect shoot total soluble sugar. HLB-exposure resulted in less negative osmotic potential, more negative turgor potential and reduced modulus of elasticity and relative water content at turgor loss point. HLB exposure also increased diurnal fruit shrinkage, with shrinkage responses to source-sink manipulation varying across fruit development stages. Collectively, these findings suggest that long-term HLB exposure promotes leaf acclimation to water deficits, however, high sink demand may diminish the leaf water status regulation at Stage II, when fruit sink carbon demand is highest.

Myricaria Desv. (Tamaricaceae) is a genus of shrubs that play a keystone role in montane riparian ecosystems across Eurasia, shaping riverine habitats and supporting ecological function. The genus has a centre of origin in the Qinghai-Tibetan Plateau and exhibits a broad distribution, but many species boundaries are unresolved. Here, we present the first phylogenomic analysis of Myricaria using the Angiosperms353 target enrichment bait set, with sampling across its full geographic range. Off-target plastid loci were extracted from next-generation sequencing data using the CAPTUS bioinformatics pipeline. We infer species relationships using ASTRAL-IV and network analysis with SplitsTree, in conjunction with morphology. We also provide a taxonomic treatment of the genus, integrating our morphological observations and phylogenomic analyses for species delimitations. Based on our analyses, recognise two infrageneric sections: sect. Parallelantherae (containing only M. elegans) and sect. Myricaria (the remainder of the genus, with type species M. germanica). We also propose synonymising M. davurica, M. bracteata and M. squamosa with M. germanica. Although species boundaries in the Asian highlands remain unresolved, we conclude that the species M. elegans, M. germanica, M. laxiflora, M. longifolia, M. platyphylla, and M. pulcherrima are well-supported both phylogenomically and morphologically.

Petal coloration is a key ornamental trait in Cyclamen persicum Mill. and is closely associated with anthocyanin composition and relative accumulation, yet the major anthocyanins and candidate molecular factors associated with color variation remain unclear. In this study, anthocyanin profiling and transcriptome analysis were integrated to investigate three Cyclamen materials with distinct petal colors, BXK, FXK, and SHXK. Anthocyanin profiling showed clear separation among the three materials, indicating that petal color divergence was associated with differences in anthocyanin composition and relative abundance. Petunidin-, peonidin-, delphinidin-, malvidin-, and cyanidin-related derivatives exhibited distinct accumulation patterns. Transcriptome analysis revealed marked transcriptional divergence, with phenylpropanoid and flavonoid biosynthesis repeatedly enriched across comparisons, whereas isoflavonoid and flavone and flavonol biosynthesis were enriched in selected comparisons. Mapping differentially expressed genes onto the anthocyanin biosynthetic pathway revealed branch-specific expression patterns of structural genes, including CHS, F3H/F3'H, F3'5'H, DFR, and ANS. Notably, Cluster-26256.0, annotated as a putative anthocyanidin synthase gene, showed a progressive increase in expression from BXK to FXK and SHXK, consistent with increasing petal pigmentation. Several DFR-related transcripts also showed relatively high expression in SHXK. Co-expression analysis linked key anthocyanins, structural genes, and transcription factors, while qRT-PCR generally confirmed RNA-seq trends. These results suggest that late anthocyanin biosynthesis, particularly ANS-related transcriptional variation, may contribute to dark red petal coloration. This study provides a metabolite-transcript framework for Cyclamen petal coloration and identifies ANS-related genes as priority candidates for future functional studies and flower-color breeding.

Extensive green roofs are an important component of urban green infrastructure. However, harsh rooftop conditions, including shallow substrates, high temperatures and limited water availability, considerably restrict plant selection. Although extensive green roofs are traditionally dominated by succulent species, increasing attention has been directed towards annual plants because of their rapid establishment, developmental plasticity and potential contribution to vegetation diversity. This review aimed to identify annual plant species introduced on extensive green roofs and to characterize the functional traits most frequently associated with their establishment under rooftop conditions. In addition, the review examined the geographical distribution and methodological characteristics of existing studies and identified species with potential for application under temperate climatic conditions. A total of 22 studies published between 2015 and 2025 were analyzed, comprising 100 annual species representing 29 plant families. The reviewed studies showed a clear geographical bias towards Mediterranean and arid regions, whereas temperate climates were considerably underrepresented. Among species occurring in Central Europe, ruderal life strategies, drought avoidance, efficient dispersal mechanisms, low plant height and prolonged flowering were the most frequently observed functional characteristics, while seed mass and specific leaf area (SLA) exhibited substantial interspecific variation. These findings suggest that functional traits, rather than taxonomic affiliation alone, provide a useful framework for selecting annual species for extensive green roofs. The review also highlights the need for long-term studies conducted under temperate climatic conditions to improve evidence-based plant selection and support the development of resilient and multifunctional green roof vegetation.

Understanding the genetic architecture of ecophysiological traits related to carbon gain and water loss is crucial to develop cultivars tolerant to abiotic stresses and that maintain productivity in the context of climate change. This can be achieved both through experiments in the greenhouse on potted plants under controlled conditions and in the field. However, the stability of genetic determinism of ecophysiological traits across environmental conditions has rarely been evaluated. We studied a grapevine diversity panel grown both in a greenhouse and in the field, to investigate the impact of growing conditions on leaf ecophysiological traits related to leaf morphology, fluorescence, gas exchange and water use efficiency. The correlation structure between traits was conserved within each environment even though, for most traits, cultivars ranking was not conserved between both environments. However, a set of cultivars, sharing the same functioning pattern in terms of gas exchange and fluorescence in both environments, was identified. Single- and multi-environment Genome-Wide Association Studies (GWAS), based on a dense genotyping matrix, were used to dissect the genetic architecture of leaf traits. The Quantitative Trait Loci (QTLs) identified through single-environment analyses differed substantially from those detected using multi-environment ones. Multi-environment approaches highlighted shared QTLs across environments for gas exchange and fluorescence traits. Candidate genes associated with carbon- and water-related functions were further identified in these genomic regions. Despite the weak correlation between greenhouse and field trait values, combining both environments enabled the identification of robust genetic regions associated with ecophysiological traits of interest.

Global change has a major impact on rivers and their associated terrestrial compartment, the riparian forest. The black poplar (Populus nigra L.), the dominant species of the European riparian forest, is considered an indicator of the biodiversity dynamics of these ecosystems, but is threatened with extinction in several parts of its range. The species is dioecious and shows wide variability in many traits, enabling it to adapt to a broad range of environmental conditions. A detailed characterization of this natural variability is therefore a key issue both for the conservation of natural populations and for biological engineering aimed at restoring riverbanks. Phenology is both an indicator of climate change and an adaptation lever for populations, particularly in perennial species such as trees. In this study, we characterized the spatial and temporal variability in flowering phenology by studying 13 natural black poplar populations along the Loire River, France. Flowering date and duration showed substantial variability both within and between populations, and males generally flowered earlier than females. Despite this variability, the ranking of individual trees' flowering dates was highly consistent across years. Most male-female pairs shared several days of overlapping flowering each year, and this overlap was moderately stable across years. Flowering dates advanced by about five to six days per degree Celsius during early spring, with no detectable difference in sensitivity between sexes. This work contributes to the characterization of flowering variability in an economically important tree species and may inform both the conservation of genetic resources and breeding.

Asian Passifloraceae occupy a distinctive evolutionary and ecological position within the family despite their relatively low species diversity compared with the Neotropics. Many Asian representatives are highly localized, small and often ephemeral climbers that persist seasonally through rhizomes, tuberous stems or dormant seeds, while considerable morphological plasticity may obscure species limits and evolutionary relationships. Xishuangbanna, southwestern China, lies at the convergence of Southeast Asian, Indian-Himalayan and southern Chinese floristic elements and represents an important regional centre of Passifloraceae diversity, with two species of Adenia and five species of Passiflora, including the recently described P. xishuangbannaensis and P. menghaiensis. We investigate the evolutionary relationships, plastome evolution and population genomics of these taxa using complete plastome assemblies, a concatenated dataset of 68 selected plastid protein-coding genes, 356 reconstructed nuclear Coding Sequences (CDS), and Genotyping-By-Sequencing (GBS) across the known wild distribution of P. xishuangbannaensis. Plastid, concatenated nuclear and nuclear coalescent phylogenies were strongly incongruent and differed from published classifications based on morphology and earlier phylogenies (ITS, ncpGS, ctyGS, ndhF and trnL-F). Gene-tree discordance and phylogenetic-network inference indicated widespread incomplete lineage sorting together with introgression. The two statistically best-supported network models placed P. menghaiensis, which showed high genetic similarity to the Indian P. napalensis, as a sister lineage involved in independent reticulation events associated with P. altebilobata, P. xishuangbannaensis and P. sumatrana. A pronounced biogeographic pattern was evident within Passiflora supersection Disemma, particularly in the P. siamica-P. cochinchinensis lineage across the Hua Line, whereas relationships among other Asian Passiflora blurred this boundary. This pattern suggests that diversification and reticulation within the Asian lineage may have overlapped temporally with the development of the biogeographic divide represented by the Hua Line. The concentration of early-diverging lineages around Xishuangbanna and the eastern Himalayan-Indian region further points to this area as an important centre in the early diversification of Asian Passiflora. Within this context, the highly localized P. xishuangbannaensis may represent either a relictual of an early Asian lineage or the surviving product of an ancient reticulate complex, contrasting with lineages such as P. siamica that subsequently expanded farther across Southeast Asia. Comparative plastome analyses revealed extensive structural and gene-content evolution within subgenus Decaloba, including pseudogenization, gene and intron losses, and repeated Inverted-Repeat (IR) expansions and contractions; substantial plastome modification also characterized supersection Disemma. Passiflora sumatrana exhibited a particularly distinctive IR expansion extending into the accD-pafII region, potentially characterizing this lineage. Adenia plastomes were generally more structurally conservative, although A. penangiana displayed an IR-boundary contraction involving the rps19-rpl2 region. Mitochondrial CDS and non-coding regions provided the best-resolved phylogenetic signal and revealed strong discordance with the chloroplast phylogeny, consistent with maternal mitochondrial inheritance and variable plastid transmission, potentially involving paternal or biparental inheritance and heteroplasmy. GBS analyses of P. xishuangbannaensis using complementary distant-reference, de novo and closely related-reference approaches showed that mapping to P. organensis provided the clearest SNP distribution and population resolution. ADMIXTURE, PCA and FST analyses revealed two strongly geographically structured populations with additional substructure and substantial differentiation (FST = 0.112). Linkage disequilibrium based demographic reconstruction using GONE2 indicated declining effective population size (Ne) toward the present in both populations, with a particularly abrupt recent decline in Pop1 and a more gradual decline in Pop2. Few candidate Self-Incompatibility (SI) genes could be confidently reconstructed, including limited recovery of highly variable docking regions. However, the intracellular region of an S-locus Receptor Kinases (SRK) contained a distinctive non-synonymous substitution predicted to alter protein configuration without major disruption of overall folding, potentially contributing to compatibility differences between populations. Together, these results reveal a complex evolutionary history combining historical dispersal, incomplete lineage sorting, reticulation, morphological plasticity, plastome structural evolution and strong population differentiation. The narrow distributions and recent demographic decline of these newly recognized Xishuangbanna taxa emphasize their conservation importance, while broader Asian taxon sampling and deeper genomic sequencing will be essential to resolve the evolutionary history of this discreet and poorly known Passifloraceae radiation.

The xylem safety-efficiency trade-off is a central paradigm in plant hydraulics, yet empirical support remains inconsistent. In this study, we highlight how this inconsistency may arise from variation in environmental and evolutionary constraints and from differences in observational scale. We analysed 38 diffuse-porous angiosperm and 24 conifer species grown in a common garden experiment to quantify the relationship between embolism resistance (P50) and specific hydraulic conductivity (Ks) across and within species, and its differences between three species groups differing in evolutionary ancestry and eco-hydrological strategy. When pooling over all species, we found evidence for a safety-efficiency trade-off both across and within species. However, this pattern was driven almost exclusively by the mesic broadleaved species. In contrast, the across-species pattern was suppressed in coniferous species due to the evolutionary constraints on efficiency posed by their tracheid-based wood anatomy, while there was no evidence for a trade-off on either level in hydrophilic broadleaved species, which evolved under conditions that pose reduced selective pressure on embolism resistance. Our results demonstrate that the safety-efficiency trade-off is not universal, but emerges only in specific ecological and evolutionary contexts, highlighting the importance of multi-scale analyses and constraint-based frameworks for interpreting plant hydraulic strategies.

Understanding floral visitor dynamics along elevational gradients is essential for assessing potential implications for pollen limitation and population genetic processes in fragmented populations of cliff-dwelling alpine endemics. Physoplexis comosa (Campanulaceae) is a chasmophytic paleoendemic restricted to vertical rock faces on dolomitic and limestone bedrock from 200 to 2,800 m a.s.l. The species exhibits a specific floral morphology with fused corolla apices, basal openings for nectar access, and secondary pollen presentation, yet its functional floral visitor assemblage has remained poorly characterized across the species' elevational range. In this pilot study, we tested a two-tier camera-monitoring setup combining action cameras for diurnal monitoring with Raspberry Pi-based systems equipped with IR illumination for combined diurnal and nocturnal recording. We conducted monitoring across five elevational sites (1,480 to 2,400 m a.s.l.) in the Eastern Alps. Recorded video material was downsampled and annotated using the Addax platform alongside manual cross-validation to record physical contact events. Preliminary results confirm the technical feasibility of continuous monitoring in steep terrain and suggest possible elevational variation in diurnal visitors, alongside nocturnal activity by Geometridae moths. Camera-based observations provided higher functional resolution and revealed a different visitor assemblage than previous eDNA metabarcoding trials. This proof-of-concept establishes a methodological foundation for forthcoming multi-day monitoring campaigns and complements ongoing studies of the mating system and population-genetic analyses.

Understanding how carbon-water coupling scales from leaves to ecosystems remains a major challenge because water-use efficiency (WUE) metrics integrate physiological, hydrological, and ecological processes. We integrated tree-ring width, triple stable isotopes ({delta}13C, {delta}18O and {delta}2H), eddy covariance (EC) observations, and a process-based model to investigate the drivers of intrinsic (iWUE) and ecosystem water-use efficiency (WUEeco) across biological scales in a mature Fagus sylvatica forest between 2004 and 2023. Modelled and isotope-derived iWUE were strongly correlated (r = 0.61, p < 0.05) and both indicated an approximately 20% long-term increase, consistent with increasing gross primary productivity. In contrast, WUEeco derived from EC was lower than model estimates and exhibited a positive trend not reproduced by the model. This divergence primarily reflected differences in biological representation and hydrological integration, as the single-species model underestimated ecosystem evapotranspiration by excluding co-occurring deciduous and evergreen species together with understory vegetation. Triple-isotope analyses showed that the long-term increase in iWUE was driven by enhanced photosynthetic assimilation rather than sustained stomatal closure, whereas {delta}18O and {delta}2H variability was mainly controlled by source-water dynamics. These findings provide a mechanistic framework for reconciling leaf- and ecosystem-scale WUE and improving predictions of forest carbon-water coupling under climate change.

Plants frequently experience recurrent drought events separated by periods of rehydration. Although drought imposes strong constraints on plant physiology, prior exposure may alter subsequent stress responsiveness through memory-based mechanisms. Here, we investigated whether recurrent drought at distinct developmental stages establishes stress memory in sugarcane and whether this response persists across vegetative propagation. Two genotypes contrasting in drought tolerance and productivity (IACCTC07-8008 and IACSP95-5000, respectively) were grown under greenhouse conditions and subjected to three drought cycles imposed either at tillering or maturation stage. Gas exchange, photochemical performance, leaf water status, primary metabolite profile, and growth traits were assessed across cycles, and vegetative propagules were subsequently evaluated under renewed drought. The first drought cycle imposed strong limitations on carbon assimilation and photochemistry in both genotypes and developmental stages. However, subsequent cycles resulted in attenuated reductions in A and g, improved intrinsic water use efficiency, and partial stabilization of PSII performance, indicating a modified stress-response trajectory. Young plants displayed earlier improvements (from the second cycle), whereas in mature plants this shift was evident mainly during the third cycle. Recurrent drought promoted sustained reorganization of amino acid, carbohydrate, organic acid and polyol metabolism alongside increased root biomass and higher relative water content during later cycles. Importantly, propagules derived from drought-conditioned plants exhibited faster recovery of photosynthetic performance and reduced cumulative physiological impairment under renewed drought, despite showing similar stress sensitivity at maximum water deficit. This persistence of enhanced recovery capacity across vegetative propagation indicates that drought-induced memory was maintained beyond the initially stressed plants. Together, our findings demonstrate that recurrent drought establishes a metabolically imprinted state in clonal sugarcane, integrating physiological adjustment, metabolic reprogramming, and whole-plant acclimation. These results highlight the potential of stress memory as a mechanism supporting resilience in perennial crops exposed to increasingly recurrent drought events.

Abstract: Picrotoxane-type sesquiterpene alkaloids, including dendrobine, are characteristic constituents of Dendrobium nobile, but the enzymes that produce their proposed skeletal precursors remain in completely characterized. Here, we combined tissue metabolite profiling with genome-guided screening and biochemical characterization of terpene synthases (TPSs). GC-MS analysis revealed variation in sesquiterpenoid profiles and dendrobine signals among tissues and developmental stages. Manual curation of the chromosome-level genome yielded 16 full-length TPS-a candidates. Functional screening in an FPP-supplying Escherichia coli system showed that three closely related genes on chromosome 1, DnTPS1~DnTPS3, produced copaborneol as their dominant product. The principal product of DnTPS1 was purified and identified by NMR spectroscopy, and a purified-enzyme assay confirmed its direct conversion of FPP to copaborneol. Fluorescence imaging in rice protoplasts and Nicotiana benthamiana leaves indicated predominantly cytosolic localization of DnTPS1. Guided by sequence comparisons and structural modeling, we tested 12 DnTPS1 variants and found that substitutions at W377 and I295 altered the relative distribution of sesquiterpene products. Phylogenetic and regional gene-order analyses placed DnTPS1~DnTPS3 in the same clade and identified related TPS candidates within corresponding genomic regions of six Dendrobium species. These findings establish that three D. nobile TPSs can form copaborneol and identify DnTPS1 residues that influence product selectivity, providing a basis for testing the proposed connection to dendrobine biosynthesis.